PROJECT SUMMARY Atherosclerosis is driven partially by pathogenic CD4+ effector T cells (Teff) reactive to antigens upregulated in the lesion (athero-antigens). Importantly, CD4+CD25+Foxp3+ regulatory T cells (Treg), a subset of T cells that function to suppress the response of Teff, have been shown to play a role in controlling atherosclerosis. Within the context of atherosclerosis an imbalance may exist between pathogenic Teff and suppressive Treg in response to athero-antigens, leading to the promotion of atherogenesis. These notions prompted us to design an anti-atherosclerosis strategy aimed at resetting the balance between Treg and Teff via increasing athero-specific Treg in vivo. Previously, mirroring the use of the conventional adjuvants for enhancing immunity, we explored the concept of tolerogenic adjuvant and designed a novel immunization strategy. In mouse models of delayed-type hypersensitivity (DTH) and spontaneous type I diabetes, we showed that immunization with peptide antigen, when performed under the influence of the immunosuppressant dexamethasone (DEX) serving as tolerogenic adjuvant (a strategy we termed suppressed immunization), caused long-term desensitization of DTH and blockade of autoimmune diabetes, respectively. We showed that such protective efficacy was associated with selective expansion of antigen-specific Treg. Encouraged by these results, we here will test our hypothesis that suppressed immunization (SI) using DEX and athero-antigens may selectively expand athero-specific Treg and reduce atherosclerosis. Under Specific Aim #1, we will determine whether SI increases the ratio of Treg to Teff at the lesion site and in peripheral blood in treated mice. To that end, male ApoE-/-/Foxp3-GFP+/+ mice (we have recently generated) will be treated with SI combining DEX with HSP60-derived peptide HP1. As controls, mice will be treated with PBS or HP1 alone. Innominate arteries from treated mice will be cryosectioned, and immunohistochemical analysis will be performed to quantify Teff (CD4+Foxp3-) and Treg (CD4+Foxp3-GFP+). Antigen specificity of Treg will be analyzed via ELISPOT with single cells prepared from the aortic arch. In addition, Teff and Treg in peripheral blood will be quantified via immunostaining and flow cytometry, and antigen specificity of the Treg will be assessed by their proliferation in response to the immunizing peptide HP1. Under Specific Aim #2, we will assess anti-atherogenic efficacy of SI in treated animals. To that end, male ApoE-/- mice with established atherosclerosis will be divided into 3 groups (n e 6). One group will be dissected before treatment to establish the baseline for lesion extent. Each of the 2 treatment groups will be treated with HP1 alone (control) or SI. Subsequently, lesions in the innominate artery will be quantified by histochemical/morphometric analysis. In addition, sections of the innominate artery will be analyzed for contents of macrophage, smooth muscle cell (SMC), and collagen. Statistical analyses will be performed on obtained data. Under Specific Aim #3, we will exploit adoptive transfer to determine whether SI-induced Treg contribute to the anti-atherogenic efficacy of SI. To that end, ApoE-/-/Foxp3-GFP+ mice will be treated with SI; 4 weeks later, Treg (Foxp3-GFP+) will be isolated from the spleen and used as donor Treg (test Treg). Treg isolated from non-treated mice will be used as control Treg. CD4+/Foxp3- cells isolated from ApoE-/- mice that have developed advanced atherosclerosis will be used as pathogenic Teff. Subsequently, pathogenic Teff will be adoptively transferred alone (control), or in combination (in various ratios) with test Treg or control Treg, into recipient ApoE-/-/Rag-2-/- mice (which are T cell- and B cell-deficient). Six weeks later, atherosclerosis lesion extent in recipients will be analyzed. Completion of these specific aims is likely to generate a safe, effective, and practical anti-atherosclerosis therapy. In addition, this study will further define the role of athero-specific Treg in controlling atherosclerosis and provide useful insights pertaining to targeting these Treg optimally.